In vivo inactivation of glycosidases by conduritol B epoxide and cyclophellitol as revealed by activity‐based protein profiling. (2nd February 2019)

• Record Type: Journal Article
• Authors: Kuo, Chi‐Lin; Kallemeijn, Wouter W.; Lelieveld, Lindsey T.; Mirzaian, Mina; Zoutendijk, Iris; Vardi, Ayelet; Futerman, Anthony H.; Meijer, Annemarie H.; Spaink, Herman P.; Overkleeft, Herman S.; Aerts, Johannes M.F.G.; Artola, Marta
• Is Part Of: FEBS journal. Volume 286:Number 3(2019)
• Page Start: 584
• Page End: 600
• Publication Date: 2019-02-02
• Subjects: activity‐based probes -- conduritol B epoxide -- cyclophellitol -- Gaucher disease -- glucocerebrosidase; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.14744
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : Glucocerebrosidase (GBA) is a lysosomal β‐glucosidase‐degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism‐based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity‐based probes to visualize catalytic pocket occupancy of candidate off‐target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α‐glucosidase were identified as major off‐targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD‐like models. Enzymes: Glucocerebrosidase (EC 3.2.1.45 ), nonlysosomal β‐glucocerebrosidase (EC 3.2.1.45 ); cytosolic β‐glucosidase (EC 3.2.1.21 ); α‐glucosidases (EC 3.2.1.20 ); β‐glucuronidase (EC 3.2.1.31 ). Abstract : In vivo target engagement of mechanism‐based glucocerebrosidase (GBA) inhibitors—conduritol B epoxide (CBE) and cyclophellitol (CP)—were examined in cultured cells, zebrafish larvae and mice by competitive activity‐based protein profiling (ABPP). This method utilizes suicide fluorescent enzyme reporter molecules to assess active site occupancy of target glycosidases by inhibitors. The in vivo targets of CBE and CP and their selectivity towards GBA were revealed.